Molecular Research on the Effect of IL10 Gene Polymorphism on Obesity Parameters in Highly Physically Active Young Men

The main aim of this study was to investigate the association between 5 polymorphisms of the interleukin 10 (IL10) gene with body composition parameters in physically active young men. A cohort of 131 male students was enrolled and the following IL10 SNPs were analyzed: rs1518111, rs1878672, rs3024496, rs3024498 and rs3024505. The subjects were divided into groups depending on obesity parameters: body mass index (BMI) and fat percentage (fat %). Statistical analysis was conducted for a single locus and haplotypes, an association between SNPs and body composition parameters were tested with four genetic models: dominant, recessive, codominant and overdominant mode of inheritance (MOI).The results showed statistically signi�cant association with BMI for CCGTA haplotype in additive model (score = -2.00, p = 0.04) and in dominant model (score = -2.30, p = 0.02). The obtained results indicate the signi�cant participation of IL10 polymorphisms in the regulation of body weight in physically active people.


Introduction
World Health Organization (WHO) currently describes overweight and obesity as abnormal or excessive fat accumulation that may impair health, as identi ed by body mass index (BMI), which relates to person's weight in kilograms divided by the square of height in meters (kg/m2).An overweight person is one whose BMI is equal to or greater than 25 whereas obese is a person for whom his BMI is equal or exceed 30, despite the age or gender.Prevalence of obesity between 1975 and 2016 nearly tripled.In 2016 according to WHO data 39% of adults aged 18 years and over and 13% of the world's adult population were overweight or obese, respectively.The main cause of obesity and overweight is an energy imbalance between calories consumed and calories expended.It results from an increased intake of energy-dense foods being rich in sugars and fat and a decrease in physical activity as a consequence of sedentary work, changes in modes of transportation or increased urbanization [1].Obesity is associated with chronic low-grade in ammation state as an physiological response necessary to restore homeostasis disrupted by environmental factors.Apart from these environmental factors, also genetic factors are of profound importance.Obesity is considered a multifactorial trait, as over 700 genes and chromosomal regions have been speci ed as having in uence in body weight and metabolic regulation where obesity is only one of the symptoms of congenital genetic disorders or polygenic, associated with the presence of polymorphisms in several genes [2].
The relationship between obesity and interleukin-mediated in ammatory process has been under investigation for many years.Balistreri et al. (2010) believe that the relationship between obesity and obesity-related in ammatory diseases can be explained by evolutionary speculation, e.g. the ability to resist hunger and elicit an effective immune response to pathogens [3].Depending on the severity of metabolic disorders, people with obesity can be divided into two categories.The rst group includes people with mild disorders and low expression of pro-in ammatory factors.On the other hand, in the second group, there is a signi cant increase in pro-in ammatory factors and we can observe in this group lot of metabolic dysfunction and disorders in processes controlling metabolism.Trayhurn and Wood (2004) proposed possible explanations of this situation.The rst possibility is the release of pro-in ammatory agents from organs other than white adipose tissue, e.g. the liver.The second theory assumes that white adipose tissue secretes factors stimulating production of in ammatory markers in the liver and other organs.The third possible explanation is that the adipocytes themselves increase production of some or most in ammatory markers [4].Cellular composition of adipose tissue de nes its secretory function.The adipose tissue consists of adipocytes, lymphocytes, macrophages, mast cells, eosinophils, broblasts and cells of the blood vessel wall.The number of individual types of cells, their phenotype and distribution determine the type of adipose tissue and the degree of obesity.Macrophages in lean mice and humans make up around 5% of the cells in adipose tissue, during obesity they constitute up to 50% of all adipose tissue cells.Type II macrophages are present in the adipose tissue of lean people, while type I macrophages appear in people with obesity.Macrophages of type II are responsible for tissue remodeling and in ammation resolution [5].'Classically activated' M1 macrophages, leads to increased expression levels of TNFα and inducible nitric oxide synthase (iNOS) and the release of nitric oxide (NO).The pro-in ammatory cytokines IL12 and IL23 are likewise produced, while synthesis of the anti-in ammatory cytokine IL10 is reduced [6].Macrophages of type II are involved in the repair of damaged tissues and prevent the development of in ammation.These macrophages have been shown to secrete signi cant amounts of IL10 with a simultaneous decrease in the synthesis of IL12 and IL23 [7].
The IL10 gene is located on chromosome 1 at 1q31-1q32, comprised of 4 introns and 5 exons anked by untranslated regions (UTR), spanning approximately 5.2 kb [8].The IL10 gene is surrounded upstream by other members of the IL10 family of cytokines IL19, IL20 and IL24, and downstream by the Mapkapk2 gene.IL10 is express in many types of cells within both the innate (including macrophages, monocytes, dendritic cells, mast cells, neutrophils, eosinophils and natural killer cells) and adaptive (including CD4 + T cells, CD8 + T cells and B cells) immune systems [2,9].Many studies have shown that polymorphisms in the coding and regulatory non-coding regions of the IL10 gene have a signi cant impact on the expression level and functionality of the cytokine IL10, which is associated with susceptibility to the development of a number of diseases based on the in ammatory state.Xia et al. (2018) showed a relationship between the rs151811 polymorphism and spastic tetraplegia in patients with cerebral palsy [10].By contrast, Shahriyari et al. (2019) showed that the same polymorphism likely plays a protective role in Behcet's disease [11].Tsilidis et al. (2009) showed the relationship between the rs3024496 and rs3024498 polymorphisms and colorectal cancer [12].Lin et al. (2017) found a signi cant association between the rs3024496 genotypes and IBD in children; and the association between the prevalence of the rs3024498 SNP genotype with IBD in adults and children [13].IL10 may also be involved in controlling of body mass.Esposito et al. (2003), tested premenopausal women with obesity (n = 50) and age-matched women (n = 50) with normal weight [8].He observed elevated circulating IL10 level in the obese group, however, low IL10 levels were observed in group with metabolic syndrome.In another study, Liu et al. (2018) observed lower IL10 levels in serum of children with obesity and hypertriglyceridemia [9].Immune mechanisms may play a potential role in the development and maintenance of obesity and only few studies have investigated the relationship between excess body weight and polymorphisms in the IL10 gene.However, the exact relation between IL10 gene polymorphisms and the risk of obesity has not been clearly de ned.
As both environmental and genetic factors are involved in the pathogenesis of obesity, environmental factors may mask genetic factors, which may be critical to the results of genetic analysis.Therefore, the aim of this study was to investigate the frequencies of polymorphisms of IL10 gene and the relationship between IL10 gene polymorphisms and the body composition parameters (fat % and BMI) in homogenous population of physically active overweight and nonoverweight young men residing under uni ed environmental conditions.

Results
Among 131 investigated individuals, 39 had BMI value exceeding 25 (OVER BMI ) and for 20 of them fat content exceeded 20% (OVER Fat ).Mean BMI in OVER BMI was 27.2 ± 1.6 and mean fat share in OVER Fat was 22.9 ± 2.4%.Signi cant differences (p < 0.05) were found between the control group CON BMI and the study group OVER BMI regarding such features as weight, BMI, fat %, BMR (Basal Metabolic Rate) and total water content in the body and values of most of these variables established for OVER BMI exceeded the values for CON BMI .No signi cant differences appeared in age and height (p = 0.42 and 0.45, respectively).
Similarly, signi cant differences were established for weight, BMI, fat%, BMR and total water content between OVER Fat and CON Fat .Only values of BMR and total water content were lower in OVER Fat than in CON Fat whereas for other variables their values in OVER Fat exceeded values for CON Fat (Table 1).Selected IL10 gene SNPs and their variations are presented Fig. 1. and in Table 2. Genotype frequencies in OVER BMI group were in Hardy-Weinberg equilibrium, although the observed rs3024498 (IL10) genotype frequencies differed signi cantly from expectations (p = 0.03).The genotypes frequency at the threshold of statistical signi cance were observed for rs1878672 and rs3024496 for the Hardy-Weinberg equilibrium in the OVER Fat group (p = 0.05 and p = 0.05 respectively) (Table 3).Genotypes IL10 rs1518111, IL10 rs1878672, IL10 rs3024496, IL10 rs3024498 were not associated with BMI (Table 4).Analysis of the link between IL10 rs3024505 and BMI showed signi cant association of this SNP with BMI values exceeding 25 (Table 5).Associations were found for the overdominant genetic model (G/G + A/A vs. A/G) with OR values = 0.39, 95% CI 0.16-0.98,p = 0.04 (Table 4).In this model the genotype A/G of rs3024505 was 2.5 times lower than G/G + A/A genotypes (p = 0.06) (Table 5).The same analysis was made for association between IL10 gene polymorphisms and fat %.The only signi cant association was found for IL10 (rs3024505) in the codominant model (G/G vs. A/G) with OR = 0.22, 95% CI 0.05-1.02,p = 0.04 and in overdominant model (G/G + A/A vs. A/G) with OR = 0.21, 95% CI 0.05-0.97,p = 0.02 (Table 6).

Discussion
In the pathogenesis of obesity both genetic and environmental factors play a signi cant role [14].Due to the extensive in uence of various environmental factors, determining the in uence of genetic factors on obesity or overweight susceptibility can be complicated.To limit the in uence of varying environmental factors, this study selected a fairly homogeneous and environmentally isolated group of individuals.The study group consisted of unrelated male military cadets based in dormitories of a military university in Warsaw.All of them have undergone tness tests both during recruitment to the military college and each year during their service, to ensure an appropriate level of physical tness.Moreover, sports activities were essential part of their education and daily routine.They usually ate their meals in the university canteen, so their habitual diet was not diversi ed.Provided meals were balanced in terms of nutritional value and energetic value and they complied the nutritional recommendation for military professionals.In study group only in 29.8% of participants values of BMI exceeding 25 were observed.Excessive storage of fat in the body exceeding 20% was observed only in 15.3% of participants.Although the size of the study group was rather small, its homogeneity allowed us to reduce the in uence of environmental factors which increased the signi cance of the presented analysis.
Excess body weight may increase the chance for the development of chronic in ammation accompanied by the secretion of many pro-in ammatory factors.It has been observed that adipose tissue in obese individuals, secreting mainly proin ammatory cytokines, i.e.TNF, IL6, leptin, visfatin, resistin, angiotensin II and plasminogen activator inhibitor 1, whereas adipose tissue of lean individuals secretes anti-in ammatory adipokines such as adiponectin, as well as transforming growth factor (TGF), interleukin (IL) 10, IL4, IL13, IL1 receptor antagonist (IL1RA) and apelin [15,16].Polymorphisms in the genes of pro-and anti-in ammatory cytokines and/or their receptors may exacerbate cytokine imbalances and thus contribute to the development or worsening of obesity [17].A wide range of pro and anti-in ammatory cytokines have been analyzed in relation to obesity [15,[17][18][19], including some information regarding IL10 [8,11,16].
Single nucleotide polymorphisms (SNPs) in cytokine genes or regions in their close proximity are considered to be important in genetic control of the production of cytokines, including IL-10.IL10 as anti-in ammatory cytokine plays an important role in the regulation of the immune system by decreasing cytokine production, inhibiting matrix-degrading metalloproteinases, and promoting the switching of lymphocytes to the Th2 phenotype [20].Furthermore IL10 exerts essential control over the biochemical parameters such as LDL, VLDL, HDL, triglycerides and glucose level [21].An increase in IL10 expression was observed in adipose tissue of obese humans and rodents, which is consistent with the ndings that obesity is associated with increased levels of circulating IL10 [8].Hence, it is important to identify and characterize the regulation of IL10 in obesity, especially as it has been shown that polymorphism of genes encoding interleukins may contribute to an increased risk of obesity [20].
The aim of this study was to investigate the frequencies of polymorphisms of IL10 gene (rs1518111, rs1878672, rs3024496, rs3024498 and rs3024505) and the relation between IL10 gene polymorphisms and the body composition parameters (fat % and BMI) in physically active young men.
Our research showed the possible relationship of the haplotype CCGTA with maintaining normal body weight, as its incidence in the CON BMI group was more than two times frequent than in the OVER BMI group (additive model p = 0.04 and dominant model p = 0.02).These data support previous reports that polymorphisms and allele variants of cytokine genes may be associated with obesity and may play an important role in body weight regulation.
Our association analysis of the IL10 gene rs3024505 polymorphism with body fat percentage was statistically signi cant in the codominant model (p = 0.04) and the overdominant model (p = 0.02).In-depth tests showed that the odds of being OVER Fat for A/G were more than 4.5 times lower than for G/G (p = 0.06) for the codominant model, and that the odds of being OVER Fat for A/G were over 4.7 times lower than for G/G-A/A (p = 0.03) in the overdominant model.
One of analyzed polymorphisms 1170 C/T rs1518111 have been previously linked to a lowering expression IL-10 mRNA, have allelic association with several other variants of IL10 polymorphisms and is an indicator of poor outcome and enhanced systemic in ammation in patients with acute coronary syndrome models were constructed with respect to the minor allele.The statistical signi cant in uence of single alleles on BMI and fat % was calculated using PearsonX 2 test with STAT.package.Haplotype analysis was performed with the haplo.statspackage.The haplo.score function was used to test the association, with magnitude and direction, between alleles combinations with BMI and with fat % under the different models of inheritance (additive, dominant and recessive).Odds ratio was calculated with haplo.ccstatistic and haplotype frequencies with haplogroup function and genotype frequencies were analysed using Fisher's exact test.IBM SPSS Statistics (version 27) was used to calculate differences between groups with t-test.The level of statistical signi cance was set at the level of p < 0.05.

Conclusions
Obtained results indicate that IL10 polymorphisms are involved in the regulation of body weight in physically active people.Analysis of the association between IL10 rs3024505 and BMI showed signi cant correlation of this SNP with BMI value over 25.What is more, statistically signi cant association with BMI was found for CCGTA haplotype (rs1518111 C > T, rs1878672 G > C, rs3024496 A > G, rs3024498 T > C and rs3024505 G > A respectively).The obtained results extend and validate the theory that in ammation-related cytokine IL10 polymorphisms in uence body weight parameters, with particularly emphasis on obesity, in physically active young men.

Declarations
Author

Figure 1 Schematic
Figure 1 Schematic representation of SNPs in the IL10 gene.SNPs positions with allelic variants (major allele>minor allele) followed by rs number are indicated.Open boxes indicate exons.

Table 1
Anthropometry and body composition of the subjects for BMI and fat %.
* statistically signi cant (p < 0.05) difference in Student T test between CON and OVER groups.BMI -body mass index.BMR -basal metabolic rate.

Table 2
Selected IL10 gene single nucleotide polymorphism and their variations.

Table 4
Association analysis of the IL10 gene rs1518111 polymorphism with BMI.

Table 7
Haplotypes distribution and its association of IL10 with BMI.